Infection Sequence and Pathogenicity of Ophiostoma Ips, Leptographium Serpens and L. Lundbergii to Pines in South Africa

Fungal Diversity

Infection sequence and pathogenicity of Ophiostoma ips, Leptographium serpens and L. lundbergii to pines in South Africa

XuDong Zhoul, Wilhelm De Beer2, Brenda D. Wingfieldl and Michael J. Wingfield2*

Tree Pathology Co-operative Programme (TPCP), lDepartment of Genetics, 2Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, 0002, Republic of South Africa

Zhou, X.D., De Beer, W., Wingfield, B.D. and Wingfield, M.I. (2002). Infection sequence and pathogenicity of Ophiostoma ips, Leptographium serpens and L. lundbergii to pines in South Africa. In: Fungal Succession (eds. K.D. Hyde and E.B.G. lones). Fungal Diversity 10: 229• 240.

Three exotic bark beetles (Coleoptera: Scolytidae), Hylastes angustatus, Hylurgus ligniperda, and Orthotomicus erosus, infest Pinus spp. in South Africa. These beetles are generally considered as secondary pests, but can also act as vectors of ophiostomatoid fungi. In South Africa, at least 12 ophiostomatoid fungi are associated with the three beetle species, of which Ophiostoma ips, Leptographium serpens, and L. lundbergii, occur most frequently. The aim of this study was to test the pathogenicity of the three fungi to pines in South Africa. Two isolates of each fungus were inoculated on various species of pines in different areas of South Africa. The inoculated fungi caused resin exudation and sapwood discoloration around inoculation points. There were significant differences in lesion length between species inoculated, times of inoculation and plantation areas. Although Ophiostoma ips gave rise to longer lesions than L. serpens and L. lundbergii, our results suggest that none of these species should be considered as serious pathogens.

Key words: bark beetles, Leptographium, Ophiostoma, pathogenicity. Introduction Three exotic bark beetle species, Hylastes angustatus (Herbst), Hylurgus ligniperda (Fabricius), and Orthotomicus erosus (Wollaston), native to Europe and the Mediterranean Basin, infest Pinus spp. in South Africa (Tribe, 1992). Hylurgus ligniperda and O. erosus are generally considered as secondary pests. Hylastes angustatus, however, is more aggressive than the other two bark beetle species, and is considered as a primary pest. This insect damages pine seedlings during maturation feeding and thus, causes significant losses in newly established pine plantations (Anonymous, 1946; Tribe, 1992).

* Corresponding author: Michael J. Wingfield; email: [email protected]

229 Bark beetles are well-known vectors of fungi, and particularly Ophiostorna and Ceratocystis spp. (Munch, 1907; Whitney, 1982; Harrington, 1988; Beaver, 1989; Wingfield et al., 1993; Paine et al., 1997; Iacobs and Wingfield, 2001). These fungi generally sporulate in the galleries of their bark beetle vectors and are either carried in mycangia, on the exoskeletons, or in the guts of the beetles (Beaver, 1989; Paine et al., 1997). The relationship between ophiostomatoid fungi and their bark beetle vectors, however, varies among different hosts, fungal species and their insect vectors (Harrington, 1993a; Wingfield et al., 1995; Paine et al., 1997). Many ophiostomatoid species cause sapstain of freshly cut wood (Munch, 1907; Lagerberg et al., 1927; Seifert, 1993). Several species are also pathogenic to plants. Ophiostorna ulrni (Buisman) Nannf. and O. novo-ulrni Brasier, which cause Dutch elm disease, have killed millions of elm trees in the Northern Hemisphere during the past century (Brasier, 1990; Brasier and Mehrotra, 1995). Three host-specific varieties of Leptographiurn wageneri (Kendrick) M.I. Wingfield, which cause black stain root disease of conifers, have led to severe losses to forestry in United States and Canada (Harrington and Cobb, 1988). Less pathogenic species such as O. minus (Hedgcock) H. & P. Sydow, L. wingfieldii M. Morelet and L. terebrantis Barras & Perry, can cause significant lesions, or even kill the trees when mass inoculated (Wingfield, 1986; Harrington, 1993b; Solheim et al., 1993). In South Africa, at least 12 ophiostomatoid species are associated with the three pine-infesting bark beetles (Zhou et al., 2001). Of the 12 species, Ophiostorna ips (Rumb.) Nannf., L. lundbergii Lagerb. & Melin and L. serpens (Goid.) M.I. Wingfield, are most frequently encountered (Zhou et al., 2001). These three species have been reported to be pathogenic to conifers in many parts of the world (Mathre, 1964; Lorenzini and Gambogi, 1976; Lieutier et al., 1989; Kaneko and Harrington, 1990; Otrosina et al., 1997). A number of preliminary pathogenicity trials have been conducted with these species on pines in South Africa (Wingfield and Knox-Davies, 1980; Wingfield and Marasas, 1980, 1983; Wingfield and Swart, 1989; Dunn et al., 2002). Little is, however, known regarding their relative importance or pathogenicity to pines in the area. The aim of this study was, therefore, to test and compare the pathogenicity of the three most frequently encountered fungal associates of pine-infesting bark beetles in South Africa. These tests were conducted on two-year-old pines representing a number of key species and in two different geographic areas. Materials and methods

Screening of fungal isolates

230 Fungal Diversity

All isolates used in this study were obtained during a survey of ophiostomatoid fungi associated with the three pine infesting bark beetle species in South Africa (Zhou et al., 2001). Fungal isolates were selected based on their relative growth rate in culture, because this was shown in preliminary trials (Wing field, unpublished) to correlate strongly with pathogenicity. Initially, 139 isolates of O. ips, 116 of L. serpens, and 138 of L. lundbergii were screened on 2% MEA (Malt Extract Agar: 20 g Biolab malt extract, 20 g Biolab agar and 1000 mL distilled water) at 25 C in the dark for two weeks. The two fastest-growing isolates of each species were chosen for the pathogenicity trials. All isolates used in this study are maintained in the Culture Collection (CMW) of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, 0002, Republic of South Africa.

Inoculation experiments Pinus spp., which are known hosts of the bark beetles and the three fungal species, were chosen for the pathogenicity trials. The availability of trees, as well as locations where the bark beetles occur, were also considered. Field inoculations were conducted in two-year-old plantations in the Western Cape province (Knysna, 23°04'00"E, 33°56'00"S) and Mpumalanga province (Sabie, 30039'00''E, 25°08'00"S), South Africa. In Knysna, two pine species, P. radiata and P. elliottii, were selected for inoculations. In Sabie, P. elliottii, and a hybrid of P. elliottii and P. caribaea, were used. Inoculum was prepared by growing fungal isolates on 2% MEA at 25 C in the dark for two weeks. After this period, cultures had commenced sporulation and the agar surface was covered with dark mycelium. Twenty trees of each pine species were inoculated with each of the six isolates. An equal number of trees of each species served as controls. One branch per tree, with an average of 20 mm diam., was inoculated. A plug of bark was removed, using a sterile 10 mm-diam. cork borer, to expose the cambium. An agar plug of equal size, bearing the test fungus, was placed mycelium side down, in each wound. Sterile agar plugs were used as controls. All inoculation points were sealed with masking tape to reduce desiccation. Six weeks after first inoculation, all trials were repeated by inoculating a second branch of the same trees inoculated during the first trial. Branches were examined six weeks after inoculation by removing the bark and exposing the cambium. Lesion lengths and branch diameters were measured. Reisolations were done by transferring pieces of freshly cut, discoloured cambium to 2% MEA. Cultures were incubated at 25 C for two weeks, after which they were microscopically examined to confirm that the lesions had been caused by the inoculated fungi.

231 Data analysis All data sets were analysed separately. Isolates belonged strictly to a specific fungal species and measurements were done on two different inoculated branches of the same tree. Therefore, a hierarchical ANOV A was employed for the analysis. The treatment variances differed somewhat, and to improve the accuracy of the ANOV A by eliminating the effect of branch diameter, branch diameter was used as a covariate in an ANCOV A. However, branch diameter was non-significant, and had no influence over lesion measurements. Differences between times of trials, species and isolates were evaluated by using a multiple comparison method adjusted to maintain the accuracy of the comparisons (Tukey - Kramer) (Anonymous, 1989).

Results

Screening of fungal isolates The fastest growing isolates of L. lundbergii (CMW6185 and CMW6186) and L. serpens (CMW6187 and CMW6188), originated from H. angustatus infesting P. patula in Mpumalanga province. Isolates of O. ips selected, however, came from O. erosus infesting P. patula in Mpumalanga province (CMW6189), and P. elliottii in Kwazulu-Natal (CMW6190), respectively.

Inoculation experiments Six weeks after inoculation, resin exudation was visible, and the inoculated fungi caused discoloration of sapwood on inoculated branches. However, no signs of dieback were seen. The branches inoculated with test fungi had more resin around inoculation points than controls. Reisolations from inoculated branches consistently yielded the inoculated fungi. Ophiostoma ips was more pathogenic than L. serpens and L. lundbergii, and generally gave rise to longer lesions. The lesion length average of O. ips from the four sites was 33.3 mm, varying between 28.7 mm and 48.5 mm. For L. serpens, lesion length average was 27.8 mm (between 15.2 mm and 44.9 mm), and for L. lundbergii, it was 29.3 mm (between 15.4 mm and 37.2 mm) (Tables 1, 2). In the Sabie area, however, O. ips (with lesion length average 31.1 mm) caused slightly shorter lesions than L. serpens (32.7 mm) and L. lundbergii (31.4 mm) (Table 2). The hybrid of P. elliottii and P. caribaea was generally more susceptible to the test fungi than P. elliottii and P. radiata. This hybrid had a lesion length average of 32.6 mm, while the average lesion lengths for P. radiata, and P. elliottii in Sabie, and P. elliottii in Knysna, were 28.9 mm, 30.1 mm, 28.9 mm,

232 Fungal Diversity

Table 1. Lesion length means (mm) for different isolates of Ophiostoma ips, Leptographium lundbergii, and L. serpens at the various inoculation sites.

Species2020of29.131.829.731.644.930.127.83134.436.236.628.4trees41.244.548.533.632.429.925.125.625.220.221.428.527.833.8P.34.328.826.921.315.228.63422.315.42Trial129.728.126.228.730.629.235.83033.727.937.224.6elliottiiSabieLesionCMW6189CMW6l90IsolateCMW6188CMW6187CMW6186CMW6l85No.lengthno.P. radiata11meanKnysna L.O.Ophiostoma* lundbergii1serpensserpenslundbergiiips= first setipsof CMW6189trials at each site; 2 =LeptographiumrepetitionP. caribaeaof the CMW6185completeP. elliottiitrialxno.*onP.theelliottiisecondL.eachbranchlundbergiitree.of

Table 2. Lesion lengths (mm) associated with inoculation of Ophiostoma ips, Leptographium lundbergii, and L. serpens onto various pine species in two geographic areas of South Africa.

Species 28.924.62834.133.327.829.331.428.932.736.531.121.42338.926.427.230.131.9Sabie32.633.5 29.629 OverallmeanP.Knysnaradiata P. elliottii Mean MeanOphiostomalundbergii ips P.30.6elliottiicaribaeax P. elliottii Mean Leptographium L.33.9serpens

respectively. Interestingly, P. elliottii in Sabie (with lesion length average 29.6 mm) was more resistant to O. ips than it was in Knysna (38.9 mm). However, P. elliottii was more susceptible to L. serpens in Sabie (31.9 mm), than it was in Knysna (21.4 mm) (Table 2). In general, longer lesion lengths were recorded in the Sabie area than in the Knysna area. In Sabie, the lesion length average was 31.4 mm, while it was 28.9 mm in the Knysna area. Ophiostoma ips, however, gave rise to a different trend. In the Knysna area, the fungus had a lesion length average of 36.5 mm, while it was 31.1 mm in the Sabie area (Table 2). Multiple comparisons of lesion length showed that there were no significant differences between the lesion lengths for the two trials with L.

233 Table 3. Comparison of the differences between lesion lengths (mm) after inoculations with Ophiostoma ips, Leptographium lundbergii, and L. serpens at two different times.

Species Index Sabie Knysna P. elliottii x P. P. elliottii P. radiata P. elliottii caribaea Leptographium D! -2.7(32.53,35.24) 3.9 (30.9, 27) 0.2 (28.1, 27.9) -3.1 (25,28.1) lundbergii p2 1.0000 0.9931 1.0000 0.9997 L. serpens D -7.9 (29.3, 37.2) -2.0 (30.9, 32.9) -6.4 (21.3, 27.7) -7.4 (17.7, 25.1) P 0.1123 1.0000 0.4476 0.1650 Ophiostoma ips D 0.2 (30.6, 30.4) -0.3 (29.4,29.7) .-10.6(28.8,39.4) -11.8 (33,44.8) P 1.0000 1.0000 0.0012 0.0001

I Difference of the lesion length means between the first and second trials; 2 Probability value; 3 Lesion length mean of the first trial; 4 Lesion length mean of the second trial.

Table 4. Combined ANOV A for lesion length measurements of the two trials at each of the four sites.

DF-Z--. SS3 MS4 FS p6 Site 3 2001.37 667.12 6.33 0.0003 Trees at each site 76 8597.14 113.12 1.07 0.3193 Species 2 5085.55 2542.77 24.13 0.0001 Site x Species 6 12382.22 2063.7 19.58 0.0001 Times! 1 3771.78 3771.78 35.79 0.0001 Species x Times 2 1487.75 743.88 7.06 0.0009 Site x Times 3 2115.42 705.14 6.69 0.0002 Site x Species x Times 6 1425.77 237.63 2.26 0.0364 ! Initial and repeated inoculations; 2 Degree of freedom; 3 Sum of squares; 4 Mean square; 5 F value; 6 Probability value.

lundbergii and L. serpens, or for O. ips in the Sabie area (Table 3). However, for O. ips, there were significant differences on the two pine species in the Knysna area (P = 0.0012, P = 0.0001) (Table 3). Combined analysis of variance (Table 4) for lesion length of the two trials at each of the four sites, showed that there are significant differences between experiment site (p = 0.0003), species (p = 0.0001), site x species (p = 0.0001), times of trials (p = 0.0001), species x times of trials (p = 0.0009), site x times of trials (p = 0.0002), and site x species x times of trials (p = 0.0364). No significant differences were found between trees at each site (Table 4).

Discussion Results of this study showed that O. ips, L. serpens and L. lundbergii can cause lesions in the cambium of Pinus spp. in South Africa. However, none of the three species inoculated caused outward symptoms such as die-back on trees. This suggests that they are weak pathogens and confirms the results of

234 Fungal Diversity previous studies where these fungi have been tested separately on a limited number of tree species (Wingfield and Knox-Davies, 1980; Wingfield and Marasas, 1980, 1983; Wingfie1d and Swart, 1989). Of the three species tested, O. ips caused the longest lesions. Leptographium serpens and L. lundbergii gave rise to similar lesion lengths, which were generally shorter than those associated with O. ipso Our results have shown that O. ips can cause lesions, but is not particularly pathogenic to pines in South Africa. This is in agreement with the studies of Wingfield and Marasas (1980), Rane and Tattar (1987), Parmeter et al. (1989), and Dunn et al. (2002). There are other studies, however, showing that the fungus was pathogenic to pines. In western Japan, O. ips, the associate of an Ips sp. infesting P. densiflora and P. thunbergii, infests the roots and has been reported to cause death of living pine trees in forests (Nisikado and Yamauti, 1933). The fungus has also been shown to significantly inhibit sapflow of infected Pinus ponderosa (Mathre, 1964). In France, it is pathogenic to Scots pines and possibly plays a role in the establishment of Ips sexdentatus (Boerner) on trees (Lieu tier et al., 1989). In the United States, O. ips, together with L. terebrantis and L. procerum (Kendrick) M.J. Wingfield, is important in the dynamics of susceptibility of southern pines to the attack by the southern pine beetle, Dendroctonus frontalis (Zimmermann) (Otrosina et al., 1997). Neither L. lundbergii nor L. serpens was pathogenic to living healthy pines in South Africa. This is interesting, since L. serpens has been recorded to be associated with a root disease of P. pinea in Italy (Lorenzini and Gambogi, 1976), and P. pinaster and P. radiata in South Africa (Wingfield and Knox• Davies, 1980; Wingfield et al., 1988). Leptographium lundbergii has been found to be weakly pathogenic to severely stressed red and black pines in Japan (Kaneko and Harrington, 1990). Ophiostoma ips, which was more pathogenic than L. serpens and L. lundbergii, is primarily vectored by the non-aggressive O. erosus (Zhou et al., 2001). The two Leptographium spp. are mainly isolated from H. angustatus, which is considerably more aggressive than O. erosus (Zhou et al., 2001). This situation, where the less aggressive bark beetle carries the more virulent fungus, has also been observed in other studies (Owen, 1987; Harrington, 1993a, b). Owen (1987) found that the more virulent fungus, L. terebrantis, was vectored by a less aggressive bark beetle, Dendroctonus valens (LeConte). There are, however, also studies indicating that more aggressive conifer• infesting bark beetle species vector more virulent fungi (Krokene and Solheim, 1998; Solheim et al., 2001). For example, Ophiostoma canum (Munch) H. & P Sydow, the major associate of Tomicus minor (Hartig), was found to be less

235 virulent than L. wingfieldii and O. minus, the main associates of T. piniperda (Linnaeus) (Solheim et al., 2001). Langstrom and Hellqvist (1993) showed that T. minor is less aggressive than T. piniperda. In our study, the hybrid of P. elliottii and P. caribaea was more susceptible to the test fungi than P. elliottii and P. radiata. Pinus elliottii was more resistant to O. ips, while more susceptible to L. serpens in the Sabie area than in the Knysna area. These results suggest that different hosts differ in their response to fungal penetration. This is in agreement with the study of Raffa and Smalley (1995), where P. resinosa and P. banksiana showed different response patterns to O. ips and O. nigrocarpum (R.W. Davidson) De Hoog. In the Sabie area, the tested fungi caused longer lesions than in the Knysna area, with the exception of O. ipso This might be explained by interactions between hosts, fungal species, climatic and other conditions in the two areas. This would be consistent with the fact that forest stand density has an influence on the infection by blue-stain fungi (Christiansen, 1985), that high water tables can increase the rate of black-stain root disease (Kulhavy et al., 1978), and that stand conditions affect the expression of host resistance (Peter and Lorio, 1993). Our results have shown that between the first and second trials, there were no significant differences in lesion length for L. serpens and L. lundbergii in the two areas, and of O. ips in the Sabie area. However, lesion lengths for the two trials using O. ips differed significantly in the Knysna area. The differences could be due to the interactions of hosts, fungi, and stand conditions, rather than seasonal difference. This is in agreement with the study of Parmeter et al. (1989), though other reports suggest seasonal difference affects the host response (Paine, 1984; Lorio, 1986). Analysis of the combined ANOV A confirmed that interactions were significant, not only between sites, times of trials, fungal species inoculated, but also between site x times of trials, site x species, species x times of trials, and site x times of trials x species. Similar results have been found by Dunn et al (2002). They showed that pathogenicity of O. piliferum (Fr.) H. & P. Sydow, O. ips, and Sphaeropsis sapinea (Fr.: Fr.) Dyko & Sutton interacted strongly with host species, location, and season. Overall, our results have confirmed that O. ips, L. serpens and L. lundbergii should not be considered as serious pathogens of above ground parts of P. elliottii, P. radiata, or the P. elliottii / P. caribaea hybrid in South Africa. But both O. ips and L. lundbergii are well-known sapstain agents on pines (Lagerberg et al., 1927; Davidson, 1935; Gibbs, 1993; Seifert, 1993; Farrell et al., 1997). Therefore, the ophiostomatoid fungi, together with their bark beetle

236 Fungal Diversity vectors, should be taken into account when disease resistant clones, or control strategies against sapstain, are developed.

Acknowledgements We thank SAFCOL, the THRIP programme of the Department of Trade and Industry, the National Research Foundation (NRF), and members of Tree Pathology Co-operative Programme (TPCP), for financial support. We thank B.E. Eisenberg for assistance with statistical analyses, as well as J. Roux and other TPCP team members for assistance in conducting the inoculation trails.

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(Received 3 December 2001; accepted 15 January 2002)

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